Limited elastase digestion of pigeon liver fatty acid synthetase with retention of all partial enzyme activities.
Pigeon liver fatty acid synthetase which contains two subunits of 240,000 daltons each has been treated with elastase. This treatment yields four protein fragments which can be separated on sodium dodecyl sulfate (SDS)-gel electrophoresis. After the subunit protein has been treated with elastase, all of the partial enzyme activities catalyzed by the complex are present, but enzyme activity for fatty acid synthesis is lost. The formation of protein fragments during proteolysis has been followed by densitometric scanning of the SDS gels. The results of these scans have suggested that (a) there are two peptide components present in the highest molecular weight band, (b) both are rapidly digested to yield the second and third largest peptides, and (c) a further cleavage of the third largest peptide gives rise to the smallest of the four major peptides. Crossed-rocket immunoelectrophoretic analysis of the four protein fragments has confirmed these conclusions and established also that the three smallest peptides are homogeneous. Each of the four peptides has been isolated by preparative SDS-gel electrophoresis, and antibody to one has been prepared. This antibody fraction immunotitrates overall fatty acid synthetase activity and immunoprecipitates the native enzyme. Immunoelectrophoresis of the four elastase-digested synthetase products against this antibody showed some cross-reactivity with a peptide that was neither the precursor nor the product of the immunogen. This cross-reacting antibody was removed by reaction with the nonrelated protein to yield antibody specific for one region of the fatty acid synthetase complex.
D000818:Animals; D000936:Antigen-Antibody Complex; D010856:Columbidae; D064429:Fatty Acid Synthases / Q000302:isolation & purification / Q000378:metabolism*; D007106:Immune Sera; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D064429:Fatty Acid Synthases; D010196:Pancreatic Elastase